Measurable residual disease (MRD) in acute myeloid leukemia (AML) is monitored through detection of leukemia-associated phenotypic protein markers (LAPMs) in bone marrow aspirates, hindering disease real-time monitoring. We explored peripheral blood (PB), extracellular vesicle (EV)-based methods for MRD monitoring. To confirm that LAPMs are present in AML-derived EVs, EVs were isolated from OCI-AML3 cells by differential centrifugation and characterized according to their size (nanoparticle tracking analysis), morphology (transmission electron microscopy) and protein cargo (proteomic analysis and Western blot). CD14 and CD33 were detected in OCI-AML3 cells and their released EVs. To select a method to isolate EVs from the PB of AML patients, three techniques were tested: size exclusion chromatography followed by ultrafiltration (SEC-UF), Total Exosome Isolation Kit (Invitrogen) and Exo-spin™ Exosome Purification Kit (Cell Guidance Systems). SEC-UF allowed EV isolation with higher purity and less aggregates than the other techniques. LAPMs were detected in those EVs, but their presence depended on the isolation method. Finally, EVs from seven AML patients’ plasma were isolated by SEC-UF. LAPMs were identified in paired samples at diagnosis and remission, with differential expression throughout disease evolution. This proof-of-concept study highlights the possibility of real-time MRD monitoring through LAPMs’ analysis in AML patient’s circulating EVs.